Abstract This application is being submitted in response to NOT-OD-19-071. Here, we propose to add Down Syndrome (DS) research objectives to our parent R01 grant, originally not focused on DS. Our new DS-focused objectives are basic science studies addressing Component 1 of the INCLUDE project in two areas. First, we will develop a novel model system to identify and study genes involved in CHD in DS patients. Second, we will perform transcriptomic profiling studies using this new model organism for CHD in DS patients. Approximately half of DS patients have CHD, but the responsible genes are not known. This poses a challenge to understanding the molecular mechanism underlying DS-CHD. Using mouse DS models, our collaborator was able to map the DS-CHD causal genes within two small loci (or linkage groups) together containing 18 genes, and furher demonstrated that CHD requires a combination of genes from both loci. However, systematic testing of all 72 possible two-gene combinations in the mouse model would be prohibitively expensive. Because early heart development is controlled by highly conserved genetic networks from flies to humans, a Drosophila- based model testing and analysis system is an ideal approach to identification of the specific gene combinations responsible for DS-CHD. We thus propose to generate and characterize Drosophila models of CHD in DS patients based on our collaborator?s discoveries and our pilot studies. We will also perform transcriptomic profiling on fly defective hearts resulting from co-expression of causal gene combination(s), and compare this data to results from mouse models and DS patient tissue sample studies. Successful completion of this supplemental project will lead to the identification of the specific gene combination causing CHD in DS patients. This is essential for understanding the molecular mechanism underlying DS-CHD and future development of precision medicine-based therapeutic approaches to prevention and treatment.